Genetic MappingandCharacterization aeruginosa …...Genetic MappingandCharacterization ofPseudomonas...

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Vol. 158, No. 3 JOURNAL OF BACTERIOLOGY, June 1984, P. 801-808 0021-9193/84/060801-08$02.00/0 Copyright © 1984, American Society for Microbiology Genetic Mapping and Characterization of Pseudomonas aeruginosa Mutants Defective in the Formation of Extracellular Proteins BENGT WRETLIND1* AND OLGERTS R. PAVLOVSKIS2 Department of Clinical Microbiology, Karolinska Hospital, S-10401 Stockholm, Sweden,1 and Naval Medical Research Institute, Bethesda, Maryland 208142 Received 14 November 1983/Accepted 9 March 1984 We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase (Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp-J near 0', with the gene order cys-59-xcp-1-proB, and loci xcp-2, xcp-3, and xcp-31 at 35', with the gene order trpC,D-xcp-31xcp-31-xcp-2-argC. Loci xcp4 and xcp41 through xcp44 were cotransducible with proA at 40'; loci xcp-5, xcp-51, xcp-52, and xcp53 were located at 55', with the gene order leu-10-trpF-met-9010- xcp-53-xcp-51xcp-511xcp-52, and xcp-6 was located at 65' to 70', between catA and mtu-9002. Nine mutations (xcp-2, xcp-3, xcp-31, xcp4, and xcp4J through xcp45) caused decreased production of extracellular enzymes. Six strains with mutations xcp-1, xcp-5, xcp-51, xcp-52, xcp-53, and xcp-6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins, such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme. The formation and release of extracellular enzymes has been studied mostly in gram-positive bacteria such as Bacil- lus spp. (6), but there is comparatively little information about gram-negative organisms in this respect. Pseudomo- nas aeruginosa produces several extracellular enzymes and toxins, and some of these proteins are of importance in the pathogenicity of Pseudomonas infections (25, 34). Most strains produce at least two proteases, designated elastase and alkaline protease (29-31). Lipase, staphylolytic enzyme, alkaline phosphatase, and phospholipase C are also found extracellularly in broth media (7, 47, 48). Exotoxin A is a ribosylating enzyme which inactivates the ribosomal protein EF-2 in eucaryotic cells (17). Previous studies with protease-deficient mutants of P. aeruginosa PAKS-1 showed (48) that the mutations caused pleiotropic effects with decreased activities of several extra- cellular enzymes; i.e., they were exoprotein deficient. Dif- ferences in phenotypic properties between mutants indicated that a number of genetic loci were involved. This study on exoprotein-deficient Pseudomonas mutants was undertaken to determine the location of chromosomal loci for regulation and release of extracellular proteins. With a few exceptions, the mutants of strain PAKS have not been amenable to genetic mapping experiments. To facilitate genetic analysis, we decided to use exoprotein-deficient mutants of the genetically characterized PAO strain of P. aeruginosa. We demonstrated in this study that a mutation in one of several loci caused phenotypically similar, pleiotro- pic effects with reduced activities of certain extracellular proteins. MATERIALS AND METHODS Bacterial strains, plasmids, and bacteriophages. Bacterial strains, plasmids, and bacteriophages used are listed in Table 1. P. aeruginosa strains were derived from PAO1 (ATCC15692). Figure 1 shows its chromosomal map (13). * Corresponding author. The strains were kindly supplied by B. W. Holloway, Monash University, Clayton, Victoria, Australia; D. Haas, Eidgenossische Technische Hochschule, Zurich, Switzer- land; and H. Matsumoto, Shinshu University, Shinshu, Japan. All strains were stored in tryptic soy broth (TSB) with 15% (vol/vol) glycerol at -70°C. To facilitate mapping, we selected a multiply marked strain (PA0222) for our experi- ments. The Xcp (extracellular protein-deficient) mutants were isolated after treatment of PA0222 with ethyl methane sulfonate as described previously (48). The mutants were isolated as protease-deficient colonies on skim milk agar plates at a frequency of ca. 10-4. Media and cultivation conditions. Unless otherwise stated, TSB and tryptic soy agar (TSA) were used. For detection of proteolytic activity, 15% (vol/vol) skim milk was added to TSA after autoclaving. Elastase, staphylolytic, and lipase activities were tested on TSA with an overlayer of TSA containing 1% (wt/vol) elastin, heat-killed cells of Staphylo- coccus aureus Copenhagen (47), and 1% olive oil emulsified with 0.1% Tween 80, respectively. For genetic experiments we used the minimal medium described by Davis and Mignioli (4) with 0.5% glucose supplemented with amino acids or other growth factors at a concentration of 0.5 mM. Stock solutions (50 mM) of amino acids and nucleotides were stored over chloroform. Selec- tion for utilization markers (benzoate [catA], mannitol [mtu], tyrosine [tyu], and proline [pru]) was done on minimal agar without citrate and glucose. The carbon source was added at a concentration of 0.1% (wt/vol). Because of the complex nutritional requirements for the Xcp mutants derived from strain PA0222, we constructed prototrophic strains carrying the xcp mutations (Table 2). These strains were obtained either by conjugation with the plasmids FP2 or R68.45 or by transduction and were used for quantitative determination of extracellular enzymes. The enzyme activities were compared to Xcp+ strains construct- ed in the same way. The following broth media were used: (i) defined protease medium according to Jensen et al. (20) for 801 on September 30, 2017 by guest http://jb.asm.org/ Downloaded from

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Vol. 158, No. 3JOURNAL OF BACTERIOLOGY, June 1984, P. 801-8080021-9193/84/060801-08$02.00/0Copyright © 1984, American Society for Microbiology

Genetic Mapping and Characterization of Pseudomonas aeruginosaMutants Defective in the Formation of Extracellular Proteins

BENGT WRETLIND1* AND OLGERTS R. PAVLOVSKIS2

Department of Clinical Microbiology, Karolinska Hospital, S-10401 Stockholm, Sweden,1 and Naval Medical ResearchInstitute, Bethesda, Maryland 208142

Received 14 November 1983/Accepted 9 March 1984

We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation ofcertain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase (Xcp mutants). Themutations were mapped on the chromosome by conjugation and transduction. The locations were xcp-J

near 0', with the gene order cys-59-xcp-1-proB, and loci xcp-2, xcp-3, and xcp-31 at 35', with the gene ordertrpC,D-xcp-31xcp-31-xcp-2-argC. Loci xcp4 and xcp41 through xcp44 were cotransducible with proA at40'; loci xcp-5, xcp-51, xcp-52, and xcp53 were located at 55', with the gene order leu-10-trpF-met-9010-xcp-53-xcp-51xcp-511xcp-52, and xcp-6 was located at 65' to 70', between catA and mtu-9002. Ninemutations (xcp-2, xcp-3, xcp-31, xcp4, and xcp4J through xcp45) caused decreased production ofextracellular enzymes. Six strains with mutations xcp-1, xcp-5, xcp-51, xcp-52, xcp-53, and xcp-6 producedcell-bound exoproteins and had defective release mechanisms. The regulation of production of alkalinephosphatase and phospholipase C is different from other exoproteins, such as elastase, but they all seem toshare a common release mechanism. Alkaline protease had separate mechanisms for regulation and release,since this protease was found in culture supernatants of all but one of the mutants, and none of the strainshad cell-bound enzyme.

The formation and release of extracellular enzymes hasbeen studied mostly in gram-positive bacteria such as Bacil-lus spp. (6), but there is comparatively little informationabout gram-negative organisms in this respect. Pseudomo-nas aeruginosa produces several extracellular enzymes andtoxins, and some of these proteins are of importance in thepathogenicity of Pseudomonas infections (25, 34). Moststrains produce at least two proteases, designated elastaseand alkaline protease (29-31). Lipase, staphylolytic enzyme,alkaline phosphatase, and phospholipase C are also foundextracellularly in broth media (7, 47, 48). Exotoxin A is aribosylating enzyme which inactivates the ribosomal proteinEF-2 in eucaryotic cells (17).

Previous studies with protease-deficient mutants of P.aeruginosa PAKS-1 showed (48) that the mutations causedpleiotropic effects with decreased activities of several extra-cellular enzymes; i.e., they were exoprotein deficient. Dif-ferences in phenotypic properties between mutants indicatedthat a number of genetic loci were involved.

This study on exoprotein-deficient Pseudomonas mutantswas undertaken to determine the location of chromosomalloci for regulation and release of extracellular proteins. Witha few exceptions, the mutants of strain PAKS have not beenamenable to genetic mapping experiments. To facilitategenetic analysis, we decided to use exoprotein-deficientmutants of the genetically characterized PAO strain of P.aeruginosa. We demonstrated in this study that a mutationin one of several loci caused phenotypically similar, pleiotro-pic effects with reduced activities of certain extracellularproteins.

MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophages. Bacterial

strains, plasmids, and bacteriophages used are listed inTable 1. P. aeruginosa strains were derived from PAO1(ATCC15692). Figure 1 shows its chromosomal map (13).

* Corresponding author.

The strains were kindly supplied by B. W. Holloway,Monash University, Clayton, Victoria, Australia; D. Haas,Eidgenossische Technische Hochschule, Zurich, Switzer-land; and H. Matsumoto, Shinshu University, Shinshu,Japan. All strains were stored in tryptic soy broth (TSB) with15% (vol/vol) glycerol at -70°C. To facilitate mapping, weselected a multiply marked strain (PA0222) for our experi-ments. The Xcp (extracellular protein-deficient) mutantswere isolated after treatment of PA0222 with ethyl methanesulfonate as described previously (48). The mutants wereisolated as protease-deficient colonies on skim milk agarplates at a frequency of ca. 10-4.Media and cultivation conditions. Unless otherwise stated,

TSB and tryptic soy agar (TSA) were used. For detection ofproteolytic activity, 15% (vol/vol) skim milk was added toTSA after autoclaving. Elastase, staphylolytic, and lipaseactivities were tested on TSA with an overlayer of TSAcontaining 1% (wt/vol) elastin, heat-killed cells of Staphylo-coccus aureus Copenhagen (47), and 1% olive oil emulsifiedwith 0.1% Tween 80, respectively.For genetic experiments we used the minimal medium

described by Davis and Mignioli (4) with 0.5% glucosesupplemented with amino acids or other growth factors at aconcentration of 0.5 mM. Stock solutions (50 mM) of aminoacids and nucleotides were stored over chloroform. Selec-tion for utilization markers (benzoate [catA], mannitol [mtu],tyrosine [tyu], and proline [pru]) was done on minimal agarwithout citrate and glucose. The carbon source was added ata concentration of 0.1% (wt/vol).Because of the complex nutritional requirements for the

Xcp mutants derived from strain PA0222, we constructedprototrophic strains carrying the xcp mutations (Table 2).These strains were obtained either by conjugation with theplasmids FP2 or R68.45 or by transduction and were used forquantitative determination of extracellular enzymes. Theenzyme activities were compared to Xcp+ strains construct-ed in the same way. The following broth media were used: (i)defined protease medium according to Jensen et al. (20) for

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TABLE 1. Bacterial strains, plasmids, and phages usedaSource orStrain Genotype reference

P. aeruginosaPAO1PAO18PA025PAO114PA0166

PA0222

PA0307PA0381PA0436PA0473

PA0503PA0905PA0949PA01670PA02376

PA06020PA06021KS902KS903KS904KS905KS906KS909KS910KS911KS921KS923KS924KS925KS926KS928KS931

P. putidaPPN1073

PlasmidsFP2FP39R68.45

RP1

pMO752

PhagesF116Lc4

G101

Prototroph, chl-2pur-66 proB64argFJO leu-10pyrD48pyrF63 leu-17

ilv-226 his-4 lys-12 met-28 trp-6 proA82

argC54leu-38 str-7 FP2+ser-3 bla436trpFl

met-9011leu-10pur-67 cys-59 thr-9001pur-136 leu-8 chl-3 rif-lmet-9020 catAl mtu-

9002 tyu-9030 nar-9011pur-67 cys-59 proB65proB65 pru-375xcp-S'xcp-3bXCp_lbxcp-31bXCp_2bxCp-4bxcp-51bxcp-52bxcp_S3bxcp-53bXCp41bxcp42bxcp_6bxcp43bxcp45bxcp44b

arg403 trp408 met404

Hg Tra Cma IncP-8Tra Cma, unclassifiedCb Nm/Km Tc Tra Cma

IncP-1Cb Nm/Km Tc Tra

IncP-1trpCDE argC Cb Nm/Km Tc

General transducingphage

General transducingphage

(14)(21)(9)

(18)Holloway

collection(9)

(19)(41)(22)

Hollowaycollection

(27)(27)(27)(9)

(38)

40a40a

This studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis studyThis study

(28)

(35)(36)(9)

(3)

(28)

(46)

(14)

a For genetic symbols, see legend to Fig. 1 and references 13 and14. xcp, Defective production or release of extracellular proteins.

b Genotype same as for PA0222 with mutagenesis as indicated.

determination of elastase and staphylolytic enzyme, contain-ing glutamate, valine, phenylalanine, and glucose; (ii) mini-mal broth (4) supplemented with 10 g of glucose and 2 g ofyeast extract per liter (MYG) for alkaline protease; (iii) MYGbroth supplemented with 10 g of Casamino Acids per liter(MCYG) for staphylolytic enzyme and elastase; (iv) MYG

broth supplemented with 5 g of tryptone per liter for lipase;(v) phosphate-deficient medium according to Torriani foralkaline phosphatase (43); (vi) phosphate-deficient mediumfor phospholipase C (1); and (vii) dialyzed TSB for exotoxinA (24). The strains were grown in flasks on a rotary table at370C for nledia i through v and at 30°C for media vi and vii.The cultures were harvested in the stationary phase ofgrowth.

Genetic techniques. Conjugation experiments were per-formed by the plate mating technique described by Stanisichand Holloway (41). Saline suspensions of donor and recipi-ent bacteria, prepared from logarithmic-phase cultures inTSB were spread over the surface of minimal agar plates.Plates were incubated at 37°C for 48 h. Colonies were thentransferred to minimal agar plates and replicated with a filterpaper replicator (23) to skim milk agar and minimal agar todemonstrate coinheritance of unselected markers. Strainswere examined for the presence of the R plasmid R68.45 onTSA or minimal agar plates containing carbenicillin orkanamycin (0.5 g/liter). The R' plasmid pMO752, whichcontained a part of the P. aeruginosa PAO chromosome,was maintained in Pseudomonas putida PPN1073 (28) andwas transferred to Xcp mutants by membrane filter mating(16). Logarithmic-phase cultures of donors and recipientswere filtered through a Millipore membrane filter. The filterwas placed on a TSA plate and incubated at 30°C for 1 h. The

xcp-6

catM

pru-375 xcp1pur767 Iccs59pB

15'~~~~~~~~~~~~~~~~90.

75'

70'

65'

60'

55'I 50' 45'

- his 4

- lys.12

xcp.5 Y argF | (pro Axcp53 1/leu-38 XCP4met 90

trp F Ileu-10 J

FIG. 1. Map of P. aeruginosa PAO chromosome (from reference13). Genetic symbols: arg, arginine requirement; bla, beta-lacta-mase production due to insertion of Tnl; cat, catechol utilization;cys, cysteine requirement; his, histidine requirement; ilv, isoleucineplus valine requirement; leu, leucine requirement; lys, lysine re-quirement; met, methionine requirement; mtu, mannitol utilization;pro, proline requirement; pru, proline utilization; pur, adeninerequirement; pyr, uracil requirement; trp, tryptophan requirement;tyu, tyrosine utilization; xcp, defective production or release ofextracellular proteins.

5' 10'

30'

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EXOPROTEIN MUTANTS OF P. AERUGINOSA 803

TABLE 2. Prototrophic recombinants of xcp mutants used fordetermination of production of extracellular proteins in brotha

P. aeru-ginosa Genotype Construction'strain

KS1001 xcp+ C, PA0381(FP2+) x KS909KS1002 xcp+ T, F116Lc4(KS906) x PA0307KS1003 xcp+ C, KS902(R68.45) x PA025KS9041 xcp-l C, PAO381(FP2+) x KS904KS9061 xcp-2 C, PAO381(FP2+) x KS906KS9062 xcp-2 T, F116Lc4(KS906) x PA0307KS9031 xcp-3 C, PAO381(FP2+) x KS903KS9032 xcp-3 T, F116Lc4(KS903) x PA0307KS9051 xcp-31 C, PAO381(FP2+) x KS905KS9052 xcp-31 T, F116Lc4(KS903) x PA0307KS9091 xcp4 C, PA0381(FP2+) x KS909KS9231 xcp-41 C, PAO381(FP2+) x KS923KS9241 xcp42 C, PAO381(FP2+) xKS924KS9261 xcp-43 C, PAO381(FP2+) x KS926KS9311 xcp44 C, PAO381(FP2+ x KS931KS9281 xcp45 C, PAO381(FP2+) x KS928KS9021 xcp-5 C, PAO381(FP2+) x KS902KS9023 xcp-5 C, KS902(R68.45) x PA025KS9101 xcp-S5 C, PAO381(FP2+) x KS910KS9111 xcp-52 C, PAO381(FP2+) x KS911KS9211 xcp-53 C, PAO381(FP2+) x KS921KS9251 xcp-6 C, PAO381(FP2+) x KS925

a Recombinants were free from the plasmids R68.45 and FP2 (i.e.,sensitive to carbenicillin and mercury chloride).

b C, Conjugation; T, transduction.

filter was immersed in saline, and the bacteria were removedby blending in a Vortex mixer and plated on selective media.TSA supplemented with 10 mg of HgCl2 per liter was used totest mercury resistance of FP2+ bacteria (42). The transduc-tion experiments were performed as described by Haas et al.(11) with the following modification. After absorption of thephage at 37°C, the cell suspension was washed twice withsaline and plated on prewarmed (42°C) minimal agar platescontaining the appropriate growth factors. The plates wereincubated at 42°C for 2 to 3 days. Transposon-facilitatedrecombination (16, 22) was used to determine the location ofmarkers in the 0' region of the chromosome. A bacterio-phage lysate (F116Lc4) of strain PA0436 (ser-3 bla436) wasused to introduce transposon TnJ into strain KS904. Theplasmid RP1 was then introduced into the KS904: :Tnl strainby membrane filter mating. Selected marker was resistanceto kanamycin (0.5 g/liter). Transposon-facilitated geneticexchange was performed by membrane filter mating.

Unless otherwise stated, at least 100 recombinants werescored for each selected marker. The mutants used in thisstudy appeared to be genetically stable; control platesshowed no reversion of xcp or auxotrophic markers.Enzyme assays. Protease activity was determined by a

caseinolytic method (49). The extent of proteolysis wasdetermined by reading the absorbancy at 280 nm of perchlo-ric acid-soluble peptides. One unit of protease activitycaused an increase of absorbancy at 280 nm of 1.0 in 30 minat 37°C. Staphylolytic activity was measured as lysis of asuspension of cells of Staphylococcus aureus Copenhagen at25°C (48). One unit of enzyme activity caused a decrease ofabsorbancy at 650 nm of 1.0 in 1 min. Substrate for lipasewas p-nitrophenyl caprylate (48), for alkaline phosphatase itwas p-nitrophenyl phosphate (43), and for phospholipase C itwas p-nitrophenyl phosophorylcholine (1). In these assays, 1U of enzyme activity hydrolyzed 1 ,umol of substrate in 1min at 25°C. Exotoxin A was measured as the ADP ribose

transferase activity (5). Since alkaline protease was alwaysproduced together with elastase and thus could not bedetermined by the caseinolytic assay, an immunologicalassay, zone immunoelectrophoresis assay (45), was em-ployed. In the zone immunoelectrophoresis assay method,proteins were determined by electrophoresis in tubular ca-nals filled with agarose gel containing antibodies. Antiserumagainst alkaline protease was produced in rabbits as de-scribed previously (49).The enzymes were assayed both in culture supernatants

and in cell lysates. The cell pellets were disintegrated byultrasonic treatment (48) and were diluted in 0.1 M Tris-hydrochloride, pH 7.4, to the original volume of the culture.The supernatants and cell lysates were not dialyzed orconcentrated before use in the assays.

Chemicals. TSB, Casamino Acids, and tryptone werepurchased from Difco Laboratories, Detroit, Mich., kana-mycin, elastin, ethyl methane sulfonate, amino acids, Tween80, p-nitrophenyl phosphate, and p-nitrophenyl caprylatewere from Sigma Chemical Co., St. Louis, Mo.; p-nitro-phenyl phosphorylcholine was from Calbiochem, San Diego,Calif.; carbenicillin was from Astra, Sodertalje, Sweden; andalkaline protease was from Nagase Biochemicals, Fuku-chiyama, Kyoto, Japan.

RESULTS

Mapping of locus for extracellular proteins (xcp-1) in strainKS904. Results of conjugation experiments with KS904 asthe recipient strain revealed a linkage between xcp-J and theauxotrophic markers ilv-226 at 7' (54% xcp+ recombinants)and his4 at 17' (30%) when the R plasmid variant R68.45was used (Fig. 1) (9, 10, 14). Selection for the other markersof KS904 (lys-12, met-28, trp-6, and proA82) gave less than15% xcp+ recombinants. FP2-mediated conjugation withPA0381 as the donor strain (starting point for chromosomalmobilization at 0') gave 9% xcp+ recombinants when ilv-226was the selected marker. The corresponding frequency inFP39-mediated conjugation was 30% (starting point at ca. 85'[14]). In reversed crosses with KS904(R68.45) as the donorstrain, no recombinants were found after selection for pur-67or cys-59 in strain PA0949. Selection for proB (PAO18) at 3'gave only 2% (2 of 138) xcp- transconjugants.

Since selection for markers in the 90' to 95' region was notpossible with KS904, using R68.45, we used transposon-facilitated recombination to investigate that part of thegenome (22). The transposon Tnl was transferred fromPA0436 (insertion bla436 at ca. 80') to KS904 throughtransduction. The R factor RP1 mobilized the chromosomein a clockwise direction, starting from the Tnl insertion. Theresults of the crosses with KS904::Tnl bla436(RP1) as thedonor and PA06021 (pru-375 proB65) and PA06020 (pur-67cys-59 proB65) are shown in Fig. 2. Linkage analysis of theseexperiments showed that the gene order was pru-375-cys-59-xcp-1-proB65-ilv-226. Thus, xcp-J was located close to0'. Cotransduction was not detected between xcp-J and themarkers pur-67, pru-375, cys-59, or proB65 (F116Lc4). At-tempts to use FP2-mediated conjugation to determine theposition of xcp-J relative to 0' were unsuccessful owing toanomalous chromosomal mobilization of FP2 in this region(40a).Mapping of xcp-2, xcp-3, and xcp-31. R68.45-mediated

conjugation experiments showed that the xcp mutations inKS903, KS905, and KS906 were located close to the markertrp-6 (trpC,D) at 35' (39). Transduction experiments withF116Lc4 showed that the xcp mutations were cotransducible

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804 WRETLIND AND PAVLOVSKIS

A 90' 95/0

pur-67 cys-59 xcp-I pro8

B

7'ilv-226

pru-375 xcp-1 p ilv-226

__494__1 81.0 20o041.0

C pru-375 xcp-1 pro 8

11.8 1075

FIG. 2. Mapping of xcp-1 (KS904) by transposon-facilitated re-combination. The donor strain was KS904::Tnl(RP1) (xcp-J ilv-226bla-436) which starts chromosome mobilization at ca. 80' in aclockwise direction. Recipient strains were PA06020 (pur-67 cys-59proB65) in A and PA06021 (pru-375 proB65) in B and C. Selectedmarkers (underlined) were cys-59 (A), proB (B), and pru-375 (C).Solid lines show linkage between selected and unselected markers;dashed lines show linkage between unselected markers. The numberof recombinants scored was 407 in (A), 200 in (B), and 255 in (C).

with trp-6. The xcp mutation in KS906 (68% xcp+ recombi-nants) was probably at a different location to those in KS903and KS905 (94% xcp+ recombinants). An independent con-firmation of the locations was obtained through the R'plasmid pMO752. This plasmid contained part of the P.aeruginosa PAO chromosome and complemented bothtrpC,D and argC mutations in PAO (27). The plasmid wastransferred from P. putida PPN1043 (arg403 trp408 met-404) to KS903, KS905, and KS906 by conjugation. Afterselection for kanamycin resistance, the xcp mutations werecomplemented in 91 to 100% of exconjugates and, afterselection for both trp-6 and kanamycin resistance, in 100%(138 colonies examined in each experiment).

TABLE 4. Conjugational and transductional analysis of xcpmutations in the proA region at 40"'

xcp+P.aeru- Geno- recoin- No.ginosa type Method binants scoredstrain (%)

KS909 xcp4 C 96 107T 74 107

KS923 xcp41 C 99 107T 42 138

KS924 xcp42 C 95 105T 80 132

KS926 xcp43 C 100 108T 93 102

KS931 xcp44 C 100 108T 82 114

KS928 xcp45 C 100 101T NRC

a The donor strain in conjugation experiments wasPA025(R68.45), and for transduction the phage was F116Lc4. Theselected marker was proA82.

b C, Conjugation; T, transduction.c NR, No recombinants found.

To elucidate the gene order, F116Lc4 lysates preparedfrom KS903, KS904, and KS906 were used to transducePA0307 (argC; Table 3). The results showed that the xcpmutations were located between trpC,D and argC and thatxcp-2 was closest to argC. To further substantiate the findingthat the xcp mutation in KS906 differed from that in the otherstrains, an F116Lc4 lysate prepared from KS9062 (xcp-2)was used to transduce KS903 (trp-6 xcp-3). Selection for trp-6 gave 24% (33 of 138) xcp+ recombinants, which demon-strated that xcp-2 and xcp-3 were in different genes. On theother hand, xcp-3 and xcp-31 gave almost identical results intransduction experiments. Thus, the deduced map order wastrp-6-xcp-31xcp-31-xcp-2-argC (11, 35).Mapping of xcp4, xcp41, xcp42, xcp43, xcp-44, and xcp-

45. Conjugation with PA025(R68.45) or PAO381(FP2+) asthe donor strains and KS909, KS923, KS924, KS926,KS928, and KS931 as the recipient strains gave 95 to 100%xcp+ recombinants after selection for proA82 (Table 4).Selection for other markers in the 7' to 35' region gave lessthan 10% xcp+ conjugants when PA025(R68.45) was thedonor, and linkage analysis showed that all xcp+ colonieswere pro'. These xcp loci were cotransducible with themarker proA82 (Table 4), with the exception of xcp45, asattempts to transduce KS928 with F116Lc4 or G101 did notyield any transductants.Mapping of xcp-5, xcp-51, xcp-52, and xcp-53. Conjugation

with PA025(R68.45) as the donor strain and selection for sixauxotrophic markers in KS902, KS910, KS911, and KS921showed no coinheritance of xcp+ Reversed R68.45-mediatedcrosses with PA025 as the recipient strain and selection for

TABLE 3. Transductional linkage between markers in the 35' area as determined by three-factor crosses with argC as the selectedmarkera

Frequency (%) of unselected markersDonor Genotype

xcp- trp xcp- trp+ xcp+ trp- xcp+ trp+

KS906 xcp-2 trp-6 65 10 0.5 24.5KS903 xcp-3 trp-6 33 6.5 0.5 60KS905 xcp-31 trp-6 31 3 0 66

Minimal crossover no. 2 2 4 2

a The recipient strain was PA0307 (argC), and the transducing phage was F116Lc4. A total of 180 to 207 recombinants were examined ineach cross.

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EXOPROTEIN MUTANTS OF P. AERUGINOSA 805

TABLE 5. Transductional analysis of xcp mutations in the 55' areaa

Frequency of xcp- recombinants with selected marker:P. aerugi- met-9011 trpE leu-10nosa strain Genotype Phage

% No. o No. % No.scored scored scored

KS902 xcp-5 F116Lc4 47 326 17 126 14 333KS910 xcp-51 F116Lc4 44 203 17 162 16 196KS911 xcp-52 G101 42 155 17 156 12 132KS921 xcp-53 F116Lc4 74 390 58 136 40 135a The recipient strains were PA0503 (met-9011), PA0473 (trpF), and PA0905 (leu-10). The gene order is met-9011-trpF-leu-10, and the

frequency of cotransduction between met-9011 and trpF was 86%, between trpF and leu-10 it was 76% with F116L (data from reference 37).

leu-10 gave 89% (KS902, xcp-5) and 85% (KS911, xcp-53)xcp+ recombinants, respectively. A small number of recom-binants from KS910 and KS921 were examined, and linkageto leu-10 was found. The results of transductional analysesare shown in Table 5. The deduced gene order was leu-10-trpF-met-9011-xcp-53-(xcp-51xcp-5I1xcp-52).Mapping of xcp-6. R68.45-mediated conjugation with

KS925 as the recipient strain gave no xcp+ recombinants.The results with KS925(R68.45) as the donor strain areshown in Table 6. The most likely location for xcp-6 wasbetween the markers catA and mtu-9002. The examination ofextracellular protease production in recombinants has beendifficult with PA02376 as the recipient. This strain was alsodeficient in the formation of extracellular proteases andprobably possessed a mutation in an xcp locus not yetidentified.

Properties of xcp mutants. The result of agar plate assaysof extracellular enzymes are summarized in Table 7, and theresults of enzyme assays in culture supernatants and disinte-grated cells are shown in Table 8. The xcp mutants weredefective in the formation of extracellular proteolytic activi-ty, elastase, staphylolytic, and lipase activities as deter-mined by agar plate assays.

All strains, except KS9281 (xcp45) produced alkalineprotease in similar amounts in the MYG. Alkaline proteasewas not produced in MCYG or the defined protease medium(20), nor was this enzyme found in the cell pellet after growthin MYG broth. Thus, the proteolytic activity found aftergrowth in the defined medium and in MCYG was consideredto be due to the elastase, although a minor part may havebeen due to a third protease (protease fraction I and proteaseI, [29, 34, 49]). We did not attempt to determine the presenceof this protease.

Strains KS904 and KS9041 (xcp-1) produced only smallquantities of extracellular elastase and no detectable staphy-lolytic enzyme, lipase, or exotoxin A. Cell-bound activitiesof elastase, staphylolytic enzyme, alkaline phosphatase, andphospholipase C were detected in contrast to the wild-typestrain.

TABLE 6. Mapping of xcp-6 by conjugationa

Recipient Selected Map Frequency No.strain marker location recombinants scored

PAO25 argF 45' 0 69PAO25 leu-10 55' 1 138PAO114 pyrD48 55'-60' 5 68PA0166 pyrF63 60' 5 138PA02376 catAl 65' 80 137PA02376 mtu-9002 70' 79 207PA02376 tyu-9030 75' 4 69

a The donor strain was KS925(R68.45) (xcp-6).

The strains carrying the mutations xcp-2 (KS906, KS9061,and KS9062), xcp-3 (KS903, KS9031, and KS9032), and xcp-31 (KS905, KS9051, and KS9052) were phenotypically simi-lar. The recipient strain in the transduction experiments(PA0307) did not produce lipase; consequently, this enzymecould not be tested in strains KS9062, KS9032, and KS9052.The activities of elastase and staphylolytic enzyme in brothwere high, comparable to the wild-type strains, in contrast tothe corresponding activites in agar plate assays. Significantcell-bound activities of exoenzymes were not detected.The mutations of 40' near proA82 (xcp4 and xcp41

through xcp45) showed different exoprotein patterns. Theelastase and staphylolytic enzyme production in MCYGbroth was comparable to that of the xcp+ strains, but theactivities of defined protease medium were lower. All strainsproduced lipase. Significant activities of cell-bound exoen-zymes were not found.The mutations in the 55' area (xcp-5 and xcp-51 through

xcp-53) produced low or undetectable activities of all exo-proteins tested except alkaline protease. They had cell-bound activities of elastase, staphylolytic enzyme, alkalinephosphatase, and phospholipase. None of the strains pro-duced significant extracellular or cell-bound lipase activity.

Strain KS9251 (xcp-6) had cell-bound alkaline phospha-tase and phospholipase. It differed from all the other mutantswith cell-bound exoenzymes since it produced extracellularelastase, lipase, and exotoxin A.

TABLE 7. Agar plate assays of extracellular enzymes producedby xcp mutantsa

P. aeru- Geno- Staphy-ginosa Ge Protease Elastase lolytic Lipasestrain enzyme

PA0222 xcp+ +++ +++ +++ +++KS904 xcp-l + + - -KS906 xcp-2 + ++ - -KS903 xcp-3 + + - -KS905 xcp-31 + + + ++ -KS909 xcp4 + + + + +++KS923 xcp41 + + + + -KS924 xcp42 + ++ + +KS926 xcp43 + + + + + +KS931 xcp44 + + + + + +++KS928 xcp45 + + + - +++KS902 xcp-5 + + - -KS910 xcp-51 + + + -KS911 xcp-52 + - - +KS921 xcp-53 + + + -KS925 xcp-6 + + + + + +++

a The wild-type strain was PA0222 (complete genotype given inTable 1). The enzyme substrates are as described in the text. + + +,Activity of wild-type strain; + +, weak activity detected as smallzones around colonies; +, activity detected after prolonged incuba-tion only; -, no detectable activity.

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EXOPROTEIN MUTANTS OF P. AERUGINOSA 807

DISCUSSION

This study has demonstrated that the genes for regulationof formation and release of extracellular proteins in P.aeruginosa were located in different parts of the chromo-some but not in the region of the chromosome in which thestructural genes for elastase and exotoxin A were found (80'-to-85' region [12, 16]), There were indications of clusteringof xcp genes in certain areas, such as 35' (xcp-2, xcp-31xcp-31); 40' (xcp4, xcp41-xcp45); and 55' (xcp-5, xcp-S1, xcp-52, xcp-53). Analysis of the phenotype properties of thestrains showed that the mutations caused pleiotropic effectsand revealed two distinct classes of mutants, tentativelydesignated class I and class II. Similar findings have previ-ously been described for protease-deficient mutants of strainPAKS-1 (48), although it has not been possible to performgenetic analysis on these mutants.

Class I mutants (strains with mutations xcp-1, xcp-5, xcp-51, xcp-52, xcp-53, and xcp-6) were defective in the releaseof exoproteins, since alkaline phosphatase, phospholipase,staphylolytic enzyme, and elastase were cell bound, incontrast to the wild-type strain. Strain KS9251 (xcp-6)showed a slightly different pattern, since it produced extra-cellular elastase, lipase, and exotoxin A. No cell-boundelastase activity was detected in this strain. However,similarities in phenotypic properties of class I mutantssuggest that the exoproteins studied here had a commonmode of release. Immunological methods will be needed todetermine whether there is a cell-bound, enzymaticallyinactive precursor form of lipase in these mutants. Some ofthe class I mutants may be related to export mnutants ofAeromonas hydrophila, which accumulated exoproteins in-tracellularly (15). The gene for P. aeruginosa phospholipasehas recently been cloned in Escherichia coli, and the enzymewas cell bound in this organism (44). On the other hand,alkaline protease seemed to have a separate release mecha-nism, since all class I strains produced this enzyme andsecreted it. Cell-bound alkaline protease antigen was notdetected in any strain.

Class II mutants (mutations xcp-2, xcp-3, xcp-31, xcp4,and xcp41 throuvgh xcp45) were defective in the formationof exoenzymes in agar plate assays. However, in brothmedia they were able to produce exoproteins. Significantcell-bound activities of exoenzymes were not detected. Onepossible explanation for their phenotypic properties is muta-tions in regulatory genes. All class II mutations mapped inthe 35'-to-40' area of the chromosome. Exotoxin A wasdetected in culture supernatants from all strains. Gray andVasil (8) described two toxin-deficient mutants which alsomapped in this area, and one of these mutations (tox-2) wascotransducible with trpC,D (94%) and was located veryclose to the xcp-3-xcp-31 loci.The exoproteins alkaline phosphatase and phospholipase

C are phosphate regulated (1, 7). All xcp mutants producedsimilar amounts of these enzymes, although class I mutantswere unable to release these and other proteins. Alkalineprotease has a separate regulation which is different fromelastase (29, 49), and its formation is inhibited in mediacontaining free amino acids, such as the Casamino Acids-containing MCYG broth and the defined protease medium.The concentration of iron in the medium is one factor that

regulates formation of elastase, exotoxin A, alkaline prote-ase, and other extracellular products (2). Several othernutritional factors not yet characterized obviously also haveregulatory functions. Strain KS928, which was defective inthe formation of elastase and alkaline protease, may possess

a mutation affecting coordinate regulation of these enzymes.The transport of proteins through the inner membrane of

bacteria has been successfully studied in E. coli (40) and onlyrecently in P. aeruginosa (26, 33). The secretion characteris-tics of E. coli make it unsuitable for studies on the release ofextracellular proteins. P. aeruginosa, Vibrio cholerae (32),and A. hydrophila (15) produce exoenzymes and have beenused for studies on secretion mechanisms in gram-negativeorganisms. P. aeruginosa has the advantage of being thebest-characterized genetically of these bacterial species andseems to be well suited for research on the release ofexoproteins.

ACKNOWLEDGMENTS

We thank Bruce Holloway, Dieter Haas, and Viji Krishnapillai forstimulating discussions, suggestions, and generous gifts of strains.We also thank Emilio Weiss, Robert DeBell, and Sam Joseph forvaluable advice and support. Expert technical assistance was sup-plied by Karin Becker.This work was supported by the Research Institute of the Swedish

National Defense, and by the U.S. Naval Research and Develop-ment Command, Research Work Unit 63750A.3M-463750D808.AB062.

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3. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H.Richmond. 1971. Properties of an R factor from Pseudomonasaeruginosa. J. Bacteriol. 108:1244-1249.

4. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichiacoli requiring methionine or vitamin B12. J. Bacteriol. 60:17-28.

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808 WRETLIND AND PAVLOVSKIS

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